For the past eight years, the University of Delaware
Howard Hughes Medical Institute’s (HHMI)
Undergraduate
Science Education program has sent undergraduate students to the
Experimental
Biology Meetings to present their research. As part of this conference,
the
American Society for Biochemistry and
Molecular Biology sponsored its 12th
Undergraduate Poster Competition in which 13 of the attending 16 UD
students
participated. Prior to this year, the students from the University had
received
more awards in this competition than students from any other college or
university. This year, in competition with about 140 other students
from over
60 institutions, UD students received first prizes in three of the four
award
categories. In addition, six other UD students received Honorable
Mention
awards. Another student was a finalist in the Undergraduate Poster
Competition sponsored by The
American Physiological Society.

2008 Award Winners from The
University of Delaware

Three of the four First Prize
winners in the 12th Annual ASBMB Undergraduate Poster Competition were
students from the University of Delaware. They are Aly Bourreza (left),
Patrick Knerr (second from right), and Krista Neal (right). All three
were also recipients of ASBMB Undergraduate Travel Awards made to the
University of Delaware's Undergraduate ASBMB Affiliate chapter. At the meetings, the University
of Delaware Undergraduate
Affiliate (UAN) Chapter of the American Society for Biochemistry and
Molecular Biology (ASBMB) received the First Annual UAN
Outstanding Chapter Awards for the North East Region and the Nation.
Aly Bourreza serves as the chapter's president.

Among the University of Delaware
Honorable Mention winners in the ASBMB Undergraduate Poster Competition
were Stevie Dreher (left), Stephanie Culver (middle), Carly Dominica
(second from right), and Andrew Harmon (right). Vivek Desai and Melissa
Warriner were also
Honorable Mention winners but are in this photo.

Mark Sausen reciving recognition
as a finalist in the American Physiological Society's Undergraduate
Poster Competition.

The
University
of Delaware group included four faculty and sixteen undergraduates.

Identifying
Biomarkers of the
Mhc Gene
to Investigate Chemosensory Discrimination

Variability
in the highly polymorphic genes of the major
histocompatibility complex (MHC) plays a fundamental role in
influencing the
odor of urinary volatiles in the common house mouse (Mus musculus). To
better
understand the mechanisms underlying MHC-dependent chemosensory
signals, it is
critical to characterize the chemical nature of MHC-dependent odorants.
Urine
samples from MHC-genotyped mice were analyzed using capillary gas
chromatography. By employing multivariate analysis and pattern
recognition
techniques on the chromatographic data, it is possible to classify mice
by
their MHC genotype and to explore which chemical compounds are
biomarkers
capable of discrimination. Soft Independent Modeling of Class Analogy
(SIMCA)
of the data was performed and indicated that the observed genotypes
were easily
distinguishable and well separated, a strong indication that specific
compounds
differentiated the genotypes. Based on the SIMCA model, a graph of the
discriminating power of

each compound
within the model was generated. The compounds that exhibited a high
level of
genotype-discriminating power were dimethyldisulfide, methyl
methylsulfenylmethyl disulfide,
Z-5,5-Dimethyl-2-ethylidenetetrahydrofuran, and
E-5,5-Dimethyl-2-ethylidenetetrahydrofuran.The structural similarity
between the
two disulfide compounds along with the similarity of the two
tetrahydrofuran
compounds suggest that there may be a biochemical explanation for the
role that
these four compounds play in differentiating mice with dissimilar MHC
genes.
The ability to qualitatively determine specific biomarkers associated
with
genetic differences has an invaluable application in the field of
genomic
medicine.

Recipient
of an ASBMB Undergraduate Travel Award,First
Place Winner in the ASBMB Undergraduate Poster Competition,
President of the outstanding Undergraduate ASBMB Affiliate Chapter

Evaluating Anti-Androgen
Resistance by In-Vitro SelectionAly Bourrezaand John
Koh,Department
of Chemistry and Biochemistry

Anti-androgens,
such as flutamide and bicalutamide (Casodex®), used alone or in
conjunction
with chemical castration, have been used in the treatment of prostate
cancer (PCa)
for decades. However, as many as 30%- 40%
of patients treated with anti-androgens acquire a therapy-resistant
phenotype
after one to three years of treatment. Furthermore, some patients
experience clinical improvement
upon
cessation of anti-androgens, a condition known as anti-androgen
withdrawal
syndrome. Anti-androgen withdrawal
syndrome has been associated with mutations to the AR that cause an
agonist
response to anti-androgens and is one of the most difficult forms of
PCa to
treat. The goal of this
project is to redesign anti-androgens to evade
molecular mechanisms that lead to anti-androgen withdrawal syndrome.
To accomplish this, novel analogs, PLM1 and
PLM6, were developed that remain antagonists towards three AR mutants
associated with anti-androgen withdrawal syndrome. In this study,
long-term growth analyses of
the human LNCaP cell line in the presence of PLM1, PLM6, or
bicalutamide have
been performed to identify potential AR mutants that would represent an
anti-androgen withdrawal phenotype. After
approximately 4- 6 weeks, resistant colonies developed and were
selected. DNA sequence data of the
resistant colonies
showed various AR mutations. PLM6
proved
to be superior to PLM1 as fewer resistant colonies were observed
compared to bicalutamide
and PLM1. This suggests that PLM6
may be effective against this form of resistant PCa. This
work is supported by the Howard Hughes Medical Institute Undergraduate
Science
Education program and the National Institutes of Health, NIDDK;
3-R01-DK054257-09.

L1
is a membrane-bound
neural cell adhesion protein involved in the development of the nervous
system.However, L1 has been implicated in
mediating
some of the invasive properties of several cancers including gliomas
and breast
cancers when proteolyzed to create a soluble ectodomain, which then
binds to
cell surface integrin receptors in an autocrine/paracrine manner.L1 has also been shown to rescue cancerous
cells from apoptosis.It was
hypothesized that polyclonal antibodies raised against a 15 amino acid
peptide corresponding
to a critical integrin binding region of human L1 would slow the
migration of
rat 9L/LacZ and human T98 G glioma cells in
vitro, whereas the peptide alone would stimulate migration.Incubation of cells in the peptide decreased
cellular migration, possibly due to competitive inhibition.The effects of the antibody on cell migration
were inconclusive.Quail QT6 cells have
been stably transfected with an L1 ectodomain-Fc fusion protein (L1-Fc)
designed to be secreted into the media.Once
purified, it will be applied to 9L/LacZ and T98 G
cells and
analyzed using automated time-lapse microscopy.Additionally,
glioma cell migration will be analyzed while
on top of QT6
cells secreting L1-Fc.Both conditions
are hypothesized to increase the migration rates of these cell lines by
mimicking the activity of endogenous L1 ectodomains.Supported by HHMI and INBRE.

During
embryonic brain development, molecular guidance cues work in concert to
transduce
signals to guide axons to their appropriate targets. Of these cues are
proteins
of the Ig superfamily, including L1 and its chick homologue,
neuron-glia cell
adhesion molecule (NgCAM). L1 and NgCAM are neuronal cell surface
membrane
proteins shown by others to accelerate axonogenesis and increase axon
elongation. In vitro bioassays were
performed in order to test the hypothesis that NgCAM expression on the
cell
monolayer will increase rate of outgrowth, axon length, and number of
axon
processes. Embryonic Day 7 optic tectum (OT) explants were first used
to study
outgrowth, yet failed to be an efficient and reproducible model.
Aggregates
were then made from dissociated OT cells and plated onto NIH3T3
monolayers that
did or did not express uniform NgCAM and onto monolayers exhibiting
patterned
NgCAM expression. The aggregates were visualized using static images
and
time-lapse microscopy by fluorescent membrane dye. Current data has
shown an
increase in both axon length and number in the presence of NgCAM,
suggesting
its crucial role as a positive axon guidance cue. Supported in part by
an HHMI Undergraduate Science Education Grant and
NIH.

PCO
arises
from
epithelial-mesenchymal transition (EMT) of lens epithelial cells
remaining
behind following cataract surgery. In cancer systems, CD44 mediates
changes
that eventually result in EMT, although CD44 function in EMT has not
been
previously studied in the lens. In the normal adult mouse lens, we
found CD44
protein and mRNA only in the lens fiber cells. CD44 null mice did not
exhibit
any qualitative differences in the timing or extent of expression of
the EMT
marker, -SMA, as compared to WT following cataract surgery suggesting
that
CD44 is not essential for EMT. But CD44 expression is highly
up-regulated in
the remaining lens epithelial cells 12 hours following the surgery.
This
suggests that it is the earliest molecular marker ever described for
lens
response to injury and may serve as a good model to understand the
early events
in the lens epithelial response to injury and the sensitization of
these cells
to later TGFβ mediated EMT. CD44 expression is known to be up-regulated
in
response to hepatocyte growth factor (HGF) mediated signaling and HGF
has been
reported to be a potent mitogen for lens epithelial cells. Future work
will
investigate the hypothesis that HGF mediated signaling up-regulates in
the lens
in response to injury leading to up-regulation of CD44 expression and
sensitization of lens cells to EMT. Funded by the Beckman Foundation,
Barry M.
Goldwater Scholarship, HHMI, and National Eye Institute.

Proteins
that function extracellularly must undergo the
process of secretion and are subject to posttranslational modifications
before cell signaling can occur. In this
study, Drosophila harboring mutations
in alg10 are characterized. alg10
encodes a glycosyltransferase that catalyzes addition of the
terminal glucose to the growing dolichol-linked oligosaccharide just
prior to its en masse transfer to nascent polypeptides. Our study of
P-element
excision alleles ofalg10
shows that this gene product is of critical importance
during Drosophila development.
Removal of both zygotic- and maternal-derived alg10
results in severe and pleiotropic embryonic deficits, demonstrating the
importance of regulated N-glycosylation during early Drosophila
development. Removal of alg10 from larval eye imaginal discs leads to a
disordered eye of reduced size. These effects may
be mediated through the Sevenless activated MAP kinase cascade, as we
find alg10
mutant eye discs display pathway gain-of-function
phenotypes. These findings indicate that regulated N-glycosylation of a
component of the Sevenless
receptor tyrosine kinase pathway is an important target of Alg10
function. Together, our data suggest that tissue
specific addition of terminal glucose to the dolichol-linked
oligosaccharide is an essential regulatory event in Drosophila
development. Sponsored by HHMI Biological Sciences Education
Program.

Hydrogels are porous,
physically rigid, three dimensional
matrices primarily composed of water which show promise as scaffolds to
aid in
tissue regeneration. The Schneider laboratory has developed a class of
β-hairpin
peptides that self-assemble into a hydrogel in response to specific
stimuli,
such as light, pH, temperature, ionic strength, and cell culture media.
The
peptide MAX8 consists of
alternating valine and lysine residues on each β-strand flanking a type
II´
β-turn (-VDPPT-) with a glutamic acid at position 15. The
kinetics
of self-assembly for MAX8
under
cellular encapsulation conditions are fast enough to result in
homogeneous
gel-cell constructs. These constructs can be prepared in a syringe and
subsequently shear thin delivered to target sites while maintaining
cellular
homogeneity. Although it has been demonstrated that MAX8
is a suitable scaffold for tissue engineering applications, this
sequence must
be prepared by chemical synthesis due to the unnatural amino acid DProline
in the turn region. In an attempt to make this material more cost
efficient for
large-scale application, a new sequence, BBH8, was designed to contain
all
natural amino acids which can possibly be overexpressed. However,
before
overexpression, BBH8 was first chemically synthesized and its bulk
material
properties characterized under physiological and cell culture
conditions. Data
show that BBH8 behaves analogously to MAX8.
Preliminary data also demonstrate that chondrocytes can be encapsulated
with
homogeneous cellular distribution, while maintaining viability in the
network.
This suggests that BBH8 is an excellent candidate for tissue
engineering. Funding
provided by NSF CHEM0348323, NIH R01 DE016386-01, and the Howard Hughes
Medical
Institute Undergraduate Science Education Program.

Phenotypic Characterization of the 7H24 Mutant in O-xylosyltransferase

Formation of
morphogenetic gradients is a fundamental mechanism of developmental
signaling.
Establishment of the gradient dictates how tissues respond to
extracellular
signals to elicit appropriate cellular responses to a secreted ligand.
Recently, we have identified a novel mutation in the
DrosophilaO-xylosyltransferase
(oxt)that encodes a key enzyme
required for heparan and chondroitin
sulfate (HS and CS) biosynthesis, named 7H24.In
Drosophila, HS has been
shown to be essential for Wingless (Wg)/Wnt, Hedgehog (Hh) and
Decapentaplegic
(Dpp)/Bone Morphogenic Protein (BMP) extracellular ligand movement and
gradient
formation. When HS is absent downstream signaling of these elemental
pathways
that drive the Drosophila and human
developmental program is disrupted. The 7H24 mutation in oxt
is a functional null and removal of maternal and zygotic oxt is embryonic lethal with terminal
segment polarity cuticle phenotypes suggesting disruption of the Wg
and/or Hh
signaling pathway. Removal of zygotic oxt causes
homozygous mutants to die as pharate adults with small
rough eyes
and thorax defects that cannot close from the pupal case. We find
deficiencies
in Wg, Hh and Dpp signaling in the mutant larval wing imaginal discs
and
evaluation of larval eye phenotypes suggests they do not arise because
of
defects in cell type specification.

1Department of Biological Sciences2Center for Translational Cancer Research at the Helen F. Graham Cancer Center3Albert Einstein College of Medicine of Yeshiva University

Regulation and expression of ERp57 in hepatocellular carcinoma

ERp57/PDIA3, a 1,25D3
binding protein with thiol-oxidoreductase activity, resides in the
endoplasmic
reticulum (ER) owing to a C-terminal QEDL. ERp57 partitions
among cell
surface, cytoplasm and nucleus with functions including 1,25D3
binding,
ion transport, and redox modulation of transcription. A tissue array
immunoscreen of normal and diseased liver sections showed dynamic
changes in
expression in both cytoplasm and nucleus. To understand
expression of
ERp57 in hepatocellular carcinoma (HCC), its steady state distribution
was
assessed in HepG2 cells using: 1) GFP (green fluorescent protein)
tagged ERp57
and, 2) subcellular fractionation. We also measured the response
to
various treatments including PMA, 1,25D3, or TNF-α.High resolution imaging demonstrates
localization of ERp57-GFP in the ER along with additional peri-nuclear
staining. A small (<5%) fraction of ERp57 also exists in the
cytosol.
Co-treatment with PMA, alone and in combination with 1,25D3
for
times from 15´ to 1 h did not reorganize EPp57-GFP. In contrast,
TNF-α
treatment of HepG2 cells moved cytoplasmic ERp57-GFP to the nucleus in
15 min.
Ongoing studies will determine if ERp57 binds to NFkB in HepG2
cells or in normal hepatocytes after TNF-a treatment.These
studies will shed light on redox regulation of transcription by nuclear
ERp57
during TNF-a signaling in
normal and transformed liver cells.Supported by HHMI and the CTCR-UD.

Within
developing tissues, the Wingless (Wg)/Wnt signaling pathway acts as a
morphogen, where the extracellular concentration of the Wg ligand
governs the
level of pathway activation in receiving cells.
Regulated downstream target activation within these receiving cells is
essential for proper tissue growth and organization during organismal
development. Our laboratory has
identified a novel highly conserved transmembrane protein in Drosophila named Sprinter
(Srt), which
is required by secretory cells for the release of active Wg ligand
(1).
The specific mechanism of Srt function is
unknown, however without it, Wg is retained and accumulates in the
cells that
produce it. Functional Wg cannot be
effectively disseminated to target cells without complex
posttranslational
processing and packaging (2). Hence, we
hypothesize that Srt functions in the secretion of active Wg ligand by
supplying a critical activity required for maturation of Wg protein in
its
route through the secretory pathway. The
focus of this research is to begin to characterize Srt function and
determine
if the mechanism of function is conserved between humans and Drosophila. Compartment
localization
studies in Srt-Wg co-transfected cells show that the majority of Drosophila and human Srt reside
in an
undefined secretory compartment and high level Srt expression causes
cells to
produce processes that appear to contain vesicles of Wg protein (Figure
1). Furthermore, through
co-immunoprecipitation we have found that Srt exerts its function by
direct
physical interaction with the Wg protein and this interaction is
conserved
across species. These results suggest
that Srt is a multifunctional protein that acts through physical
interaction
with Wg and perhaps promotes cellular morphological changes that
support the
dissemination of functional Wg from secretory cells. This data also
indicates
that these functions are conserved across species. Furthermore, these
experiments provide the foundation for evaluating Srt function and
conservation
in developing Drosophila.

Peptides
have
been designed which undergo intermolecular self-assembly to form rigid,
fibrillar hydrogel networks. These
peptides are unstructured and soluble in aqueous solution, but can be
triggered
to fold into an amphiphilic β-hairpin conformation under specific
environmental
conditions. Once folded, these hairpins
self-assemble to form the hydrogel. Introduction of metal-binding
functionality into such a
system allows
for the development of materials in which hydrogelation can be
controlled with
particular metal ions. This work reports
a β-hairpin peptide incorporating two strategically-placed cysteine
residues,
known to bind a host of metal ions including arsenic and zinc.
Equimolar addition of methylarsonous acid to
an aqueous peptide solution triggers folding and self-assembly at low
peptide
concentration (1.0 wt %). Metal-dependent behavior has been
demonstrated using
circular dichroism,
FTIR, TEM and oscillatory shear rheology. It is envisioned that
this system could be responsive to
other
cysteine-binding toxic metals, such as mercury and lead. Since
the peptide sequence includes only
natural L-amino acids, this peptide can potentially be synthesized
biologically
and used for large-scale bioremediation of polluted environments.
Funding has been supplied by the Beckman
Foundation, the HHMI Undergraduate Biological Sciences Education
Program and
the NIH.

PTH
enhances the
mechanical loading-indiced increases in bone formation that we
postulate results from PTH disruption of the actin cytoskeleton to
increase the mechanosensitivity of the osteoblast. Since Ras homologous
A (RhoA) GTPase activation promotes action assembly, we examined the
effects of these stimuli on RhoA activation, actin organization, and
cellular stiffness. Fluid shear (12 dynes/cm2) increases
actin stress fiber formation (ASFF) within 15 min in MC3T3-E1
ostepoblasts that is blocked by pretreatment with 50nM PTH. Using a
RhoA G-LISA assay, we found that shear activated RhoA within 15
min of the onset of shear and that 15 min pretreatment with PTH
significantly inhibited this activation. AFM quantification of the
stress-strain relationship of MC3T3-E1 cells indicated that shear
produced a 6-fold increase in cellular stiffness that was blocked by
PTH pretreatment. Inhibition of RhoA-dependent kinase (ROCK), an
effector protein of RhoA with Y27632 blocked the shear-induced ASFF as
well as the increase in cellular stiffness. These changes in cell
stiffness closely correlated with shear-induced calcuim signaling.
These studies suggest that PTH inhibits shear-induced actin
organization through the RhoA-ROCK opathway and that the resultant
changes in cell stifness play a role in the PTH-mediated enhancement of
osteoblast mechanosensitivity. Supported buy an HHMI Undergraduate
Science Education Grant and NIH/NIAMS AR043222.

Eukaryotic
DNA replication is a vital molecular
process which is not fully understood. Simian Virus 40 provides an
ideal model
system. Its genome encodes the protein large T-Antigen (T-Ag), which
orchestrates the initiation of replication. T-Ag interacts with host
cellular
protein Topoisomerase I (Topo I). Topo I binds a T-Ag monomer at two
sites, one
near the N-terminal domain (residues 83-160) and one at the C-terminal
end
(residues 602-708). Preliminary data suggest that T-Ag's J-domain and
hinge
region residues 101-109, may be involved in Topo I binding. We
generated single
point mutations in full-length T-Antigen and in a N-terminal construct
that
contains residues 62-262. In vitro replication assays identified three
mutants,
N99S, L103Q, and C105F to be replication deficient. Furthermore, DNA
replication was not stimulated by the addition of Topo I, as is WT
T-Ag. ELISAs
showed mutant T-Ags N99S, E100A, L103Q, F104V, and E107G have a
decrease in
Topo I binding as compared to the WT fragment. These data indicate that
this
region is important for Topo I binding and residues N99 and L103 may be
necessary for DNA replication. Other assays will be performed to verify
that
the mutants' deficiency in DNA replication is due to a defect in Topo I
binding
and not to other causes. This work is funded by a PHS Grant from the
National
Cancer Institute to DS and a HHMI Undergraduate Award to KN.

Finalist
in the American Physiological Society's Undergrauate Poster Competition

Vice president of
the outstanding Undergraduate ASBMB Affiliate Chapter

Expression and
characterization of Loop- and Helix-forming epitopes of Measles Virus
Hemagglutinin in plants

The
number of cases of acute
Measles Virus (MV) in the United States plummeted 98%
since 1963, when a live
attenuated vaccine has been introduced. However, such a vaccine
requires
constant refrigeration to preserve potency. More importantly, the
presence of
maternal antibodies interferes with effectiveness of the vaccine before
~9
months of age. These reasons present a major impediment for the
vaccination of
young children, especially in developing countries. Here, we report the
transient expression of two MV hemagglutinin epitopes in Nicotianabenthamiana.
These loop- (L) and helix- (H) forming epitopes are not recognized by
maternal
antibodies, have been shown to generate a protective immune response in
mice,
and could be used to develop a vaccine for infants before the age of 9
months.
The epitopes were fused to the surface loop of the thermostable enzyme
of Clostridium
thermocellum, and produced in plants through a combination of viral
and
bacterial vectors. Purified antigens were characterized by SDS-PAGE
followed by
immunoblotting. Immunological testing showed that the L- and H-epitopes
are
recognized by L- and H-specific antibodies, respectively, thus
successfully
mimicking natural antigenic properties.

Targeted
nucleotide exchange (TNE) relies on the use of a short synthetic
oligonucleotide (ODN) designed to be
complementary to atargetsequenceinthegenomeexceptforacentrallylocated mismatch,
which
directs the desired base
change in the DNA.The repairreactionisbelievedtobeenhancedbymechanismsthat promote
a more open conformation of the DNA, thus, enabling the target
site to be more accessible for the binding
of the oligonucleotide.One such method is through the modulation of cell
cycle progression to increase the population of cells in S phase,
wherein
targeting of a single base pair is enhanced.Studies
aimed at demonstrating the variations in
correction in relation
to cell cycle measured repair frequency late in the reaction cycle.Our work aims to discern variations in
repair
levels in sub-phases of S as cells enter and progress through this
phase, in
the presence of the oligonucleotide.In
addition, we evaluate gene expression patterns
ofseveral cellcycle
genes that may help denote the changes
incell cycle distributionasa
function of generepair events.Additional research has been performed
analyzing gene
expression
patterns in malignant versus non-malignant esophageal cancer cells in
response
to treatment with G-rich oligonucleotides (GRO).It has been shown that these
single-stranded DNA molecules
which are comprised
primarily of guanosine residues induce retardation in the progression
of the
cell cycle, and result in asub-G1
population ofapoptotic cells.Successful inhibition of growth of
malignant, but not nonmalignant, cells has been seen.Thus,
G-rich ODNs may be guiding the selective killing of tumor cells.I am currently analyzing the RNA levels of
apoptosis genes incubated with GROs to understand how the cells are
responding,
and which pathway may be responsible for the induction of apoptosis.Funded by Howard Hughes Medical Institute.